The present invention refers to a cross-linking process of carboxylated polysaccharides.
The process of the invention provides a high degree of reproducibility of the obtained products, in terms of cross-linking degree, homogeneity of the distribution of the cross-linking chains, and chemico-physical characteristics of the products and the technological characteristics of the articles obtained therefrom.
The reproducibility is particularly important for the applications in the medical, pharmaceutical and dermo-cosmetic fields.
The invention further refers to the products obtainable by said process and their applications in the medical, pharmaceutical and dermo-cosmetic field.
The use of macromolecules in the medical/pharrnaceutical field and, more recently, in the dermatological-cosmetic field, is well established. Macromolecules are used in the preparation of pharmaceutical formulations as thickening agents, lubricants, gastro-resistant film coating agents, particularly in the preparation of capsules, gel, colloids and of different devices (e.g. contact lenses, gauzes, etc.). Macromolecules are also used in the preparation of controlled-release formulations of active ingredients.
Reviews of their characteristics and pharmaceutical uses are reported in
1) C. Hansch et Al. Editorsxe2x80x94xe2x80x9cComprehensive Medicinal Chemistryxe2x80x9dxe2x80x94Pergamon Press, Oxford, 1990xe2x80x94Vol. 1-6;
2) A. Wade and P. J. Wellers Editorsxe2x80x94xe2x80x9cHandbook of Pharmaceutical Excipientsxe2x80x9dxe2x80x94Ed. 1994xe2x80x94The Pharmaceutical Press.
Said macromolecules belong to different chemical families and may be either synthetic, or natural or semi-synthetic.
Examples of synthetic macromolecules include polyvinylpyrrolidone, polyoxyethylenealkyl ethers, polyvinyl alcohols, polymethacrylates. Examples of natural macromolecules include native hyaluronic acid (HY) and cellulose.
Examples of semi-synthetic macromolecules include carboxyalkylcelluloses, widely used in the food and personal care industries. These macromolecules are characterized by a linear or poorly branched structure.
A very important modification for increasing the chemical, enzymatic and mechanical strength is provided by cross-linking, which may be carried out both on synthetic and natural (more or less already modified) polymers.
Examples of cross-linked polymers include polymers used for the gastro-protection of tablets or capsules (polymethacrylates), as well as polymers used as emulsifiers, suspending agents, tablet hardeners (Carbopol), cross-linked hyaluronic acids, hereinafter discussed.
For the considered applications, and particularly for the preparation of invasive medical devices which have to be administered parenterally, said polymers must meet a number of requirements, of technical and regulatory kind.
The technical requirements include:
1) high biocompatibility;
2) resistance to enzymatic systems, both tissular or plasmatic (for injectable compositions) and gastrointestinal (for oral compositions).
In some cases a gradual degradation, for instance for the controlled release of a medicament, may be desirable.
This resistance is particularly important when the macromolecule is present in compositions/articles that must last for a long time, e.g. substitutes of the synovial liquid, films, sponges or gels as tissular antiadhesives in different kinds of surgery; in tissular engineering (artificial organs); artificial skins, in the treatment of burns and generally in aesthetic surgery;
3) moldability into different shapes (gels, films, sponges, etc.);
4) possibility to be sterilized chemically or physically without changing the product structure.
According to the regulatory requisites, the composition of the different production batches must be kept constant within very narrow limits; this implies that the production methods are standardized and that the base components have a very low intrinsic variability.
A possible cause of dishomogeneity for macromolecules derives from the dispersion of molecular weights. Said dishomogeneity becomes even higher as a consequence of cross-linking. This may be a serious drawback depending on the field of use and the applicative purposes of the final product.
EP-A-566118 (Kimberly-Clark) discloses cross-linked polysaccharides to be used as super-absorbents for diapers and similar articles.
The process described therein is based on the cross-linking of cellulose by formation of intermolecular amides, esters or ethers between polyamines, polyols or mixtures thereof and the carboxy group of polysaccharides.
The reaction is carried out by heating at about 80xc2x0 C. the mixture of the polysaccharide with the polyol and/or polyamine. This process is certainly economic and suitable for large scale production where the reproducibility requirements are less stringent.
U.S. Pat. No. 5,465,055 discloses cross-linked polysaccharides (hyaluronic acid and alginic acid) obtained by esterification of COOH of the polysaccliaride and OH groups of other molecules, without insertion of cross-linking arms.
WO 91/9119 discloses microcapsules for islets of Langerhans as biohybrid organs, consisting of alginic acid cross-linked with barium ions.
EP 190215 discloses the cross-linking of different polymers (carboxylated starches, dextran, celluloses) with di- or poly-functional epoxides.
The following cross-linking agents for hyaluronic acids have been proposed:
polyfunctional epoxides are disclosed in U.S. Pat. Nos. 4,716,224, 4,772,419, 4,716,154;
polyalcohols are disclosed in U.S. Pat. No. 4,957,744;
divinylsulfone is disclosed in U.S. Pat. Nos. 4,605,691, 4,636,524;
aldehydes are disclosed in U.S. Pat. Nos. 4,713,448 and 4,582,865;
carboxamides are disclosed in U.S. Pat. No. 5,356,833;
polycarboxylic acids are disclosed in EP-A-718312.
The invention refers to a process for the preparation of cross-linked polysaccharides containing carboxy groups, allowing complete control of cross-linking degree as well as high reproducibility in terms of constant characteristics of the final product.
The process of the invention comprises:
a) activation of the carboxy groups of the polysacchatide by reaction with suitable carboxy activating agents in anhydrous aprotic solvent;
b) reaction of the carboxy activated polysaccharide with a polyamine.
The obtained cross-linked polysaccharide, if desired, may be subjected to sulphation or hemisuccinylation of the free hydroxy groups.
The products obtainable by the process of the invention may also be complexed with metal ions such as zinc, copper or iron ions.
The carboxy-containing polysaccharide which may be used according to the invention may be of natural, synthetic or semi-synthetic origin. Examples of said polysaccharides include Hyaluronic acids (obtained from tissues or bacteria), carboxymethyldextran, carboxymetbylcellulose, carboxymethyl-starch, alginic acids, cellulosic acid, N-carboxy-methyl or butyl glucans or chitosans; heparins with different molecular weights, optionally desulphated and succinylated, derrnatan sulphates, Chondroitin sulphates, heparan sulphates, polyacrylic acids.
Hyaluronic acids, carboxymethylcellulose, heparins, alginic acids and polyacrylic acids are particularly preferred.
Said cross-linked polymers, obtained by different methods, are known and have been proposed for several uses (see, for instance, EP 566118, WO91/9119, U.S. Pat. Nos. 5,465,055, EP 190215, EP 718312, U.S. Pat. Nos. 4,716,224 discussed above).
The carboxy activating agents are usually those used in the peptide chemistry: examples of suitable agents include carbonyldiimidazole, carbonyltriazole, chloromethylpyridylium iodide (CMP-J), hydroxybenzotriazole, p-nitrophenol p-nitropheriyltrifluoroacetate, N-hydroxysuccinimide and the like. The use of chloromethylpyridylium iodide is particularly preferred.
The polyamines have preferably the following general formula:
R1xe2x80x94NHxe2x80x94Axe2x80x94NHxe2x80x94R2
wherein R1 and R2, which are the same or different, are hydrogen, C1-C6 alkyl, phenyl or benzyl groups, A is a C2-C10 alkylene chain, preferably a C2-C6 alkylene chain, optionally substituted by hydroxy, carboxy, halogen, alkoxy, amino groups; a polyoxyalkylene chain of formula
[(CH2)nxe2x80x94Oxe2x80x94(CH2)n]m
wherein n is 2 or 3 and m is an integer from 2 to 10; a C5-C7 cycloalkyl group; an aryl or hetaryl group, preferably 1,3 or 1,4-disubstituted benzene. A is preferably C2-C6 linear alkylene or a chain of formula
[(CH2)nxe2x80x94Oxe2x80x94(CH2)n]m
The cross-linking reaction is preferably carried out in a solvent selected from tetrahydrofuran, dimethylformamide or dimethyl sulfoxide, and the polysaccharide is preferably salified with a lipophilic cation, for example tetralkylammonium or other lipophilic organic bases.
The transformation of inorganic salts such as sodium salts, into suitable organic lipophilic salts may be carried out by known ion-exchange methods in homogeneous phase or by precipitation of the acidic component, followed by recovering of the latter and salification with the desired organic base.
The activation reaction of the carboxy groups is carried out in homogeneous phase and in anhydrous polar aprotic solvent.
The polyamine diluted in the same anhydrous solvent, is added to the solution of the activated ester, keeping the temperature from 0xc2x0 C. to 30xc2x0 C. The cross-linking reaction times range from 1 to 12 hours, also depending on the optional presence of suitable basic substances (e.g. triethylamine).
Generally, the final product is recovered by precipitation of the organic salt adding a different solvent to the reaction solvent or by evaporation of the latter, followed by centrifugation, washing with distilled water, repeated dispersions in the solutions of the desired alkali (for instance sodium, potassium), subsequent washing with water and final drying of the alkaline salt under vacuum or by lyophilization.
The cross-linking degree (C.L.D) may range within wide limits and may be adjusted by changing the amount of the carboxy activating agents, since the activation and the cross-linking reaction are substantially quantitative.
The cross-linked polysaccharides obtained according to the invention may be subjected to sulphation reaction of the hydroxy groups possibly present, usually by reaction with the pyridine-sulfur trioxide complex in dimethylformamide.
The reaction is carried out in heterogeneous phase at a temperature of 0-10xc2x0 C. for times ranging from about 0,5 to about 6 hours.
The sulphation degree obtained is comprised within wide limits with respect to the total of the hydroxy groups and it may be adjusted by changing the temperature and reaction times. Generally, the sulphation degree (defined as equivalents of sulphate groups/g) may range from 1xc3x9710xe2x88x926 to 6xc3x9710xe2x88x926, preferably it is of 2xc3x9710xe2x88x926 eq/g for a cross-linking degree of 0.5.
The cross-linked polymers obtained according to the invention, optionally sulphated, are able to complex metal ions such as zinc, copper or iron ions.
Said complexes may be obtained by dissolving or dispersing until complete swelling the product in water and adding under stirring, preferably at room temperature, a concentrated solution of an organic or inorganic metal salt, e.g. CuCl2, ZnCl2, Fe2(SO4); after stirring for 12-24 hours, the complex is recovered by centrifugation or by precipitation following the addition of a different solvent (for example ethanol or acetone) or evaporation under vacuum; the recovered crude product is thoroughly washed with distilled water so as to remove the excess ions. The complexes are then lyophilized. The content of metal ions varies depending on the used operative conditions, particularly the polymer to ion molar ratios; concentration and pH of the solutions; reaction times and particularly cross-linking degree.
The process of the invention, by suitably adjusting the cross-linking and/or sulphation degree, allows the preparation of cross-linked carboxylated polysaccharides in a wide range of shapes, characterized by different properties such as viscoelasticity, hydration degree, complexing ability towards metal ions, ability to form hydrogels, moldability in films or sponges, mechanical strength of the final materials.
This allows their use in many medical fields, in the human and veterinary field, and in dermo-cosmetology.